Chromatin, located in the nucleus of eukaryotic cells, is the complex of DNA with histone proteins, including, H1, H2A, H2B, H3, and H4. The histone proteins are assembled onto DNA in the form of a nucleosome (Luger et al. (1997) Nature, 389, 251-260). The DNA sequence carries the genetic code and controls inheritance of traits. However, reversible covalent modifications to specific DNA sequences and their associated histones can influence how the underlying DNA is utilized and can therefore also control traits (Jenuwein and Allis (2001) Science, 293, 1074-1080; Klose and Bird (2006) Trends In Biochemical Sciences, 31, 89-97). These have been referred to as epigenetic modifications. The most common epigenetic modifications to DNA in mammals are methylation and hydroxymethylation of DNA, either of which may be placed on the fifth carbon of the cytosine pyrimidine ring. A host of modifications including methylation, acetylation, ribosylation, phosphorylation, sumoylation (related to small ubiquitin-like modifiers), ubiquitylation and citrullination can occur at more than 30 amino acid residues of the four core histones within the nucleosome. Epigenetic modifications to the mammalian genome include methylation of cytosines in the DNA; methylation, acetylation, ribosylation, phosphorylation, sumoylation and ubiquitylation of the histones bound to the DNA; and the precise positioning of histone containing nucleosomes over the DNA.
Epigenetic alterations to the genome can influence development and health as profoundly as mutagenesis of the genome. One of the most dramatic examples is the methylation of DNA at the promoter of the p16 tumor suppressor. Such methylation silences the gene, and so do mutations to the gene sequence itself, and both events contribute to the development and progression of colorectal cancer. However, unlike mutations, epigenetic silencing of p16 can be reversed pharmacologically, and hence providing the possibility of medical intervention.
Specific changes in epigenetic state that occur genome wide appear to regulate cellular differentiation during development (Mikkelsen et al. (2007) Nature, 448, 553-U552). Perturbations of normal epigenetic state in mature tissues contribute to initiation and progression of cancer and other diseases (Feinberg, A. P. (2007) Nature, 447, 433-440). Additionally, studies have shown that epigenetic states are influenced by environmental variables including diet (Waterland and Jirtle (2003) Molecular And Cellular Biology, 23, 5293-5300), environmental toxins (Anway et al. (2005) Science, 308, 1466-1469) and maternal behaviors (Weaver et al. (2004) Nature Neuroscience, 7, 847-854). Given the fundamental role that epigenetic mechanisms play in normal development, environmental responses and how their perturbation affects disease state, there is increasing effort devoted to characterizing the human epigenome (Bernstein et al. (2007) Cell, 128, 669-681).
These epigenetic modifications do not alter the primary DNA sequence, but they have a potent influence on how those underlying DNA sequences are expressed. As a result, changes in epigenetic state can alter phenotypes as powerfully as alterations in DNA sequence. Also like DNA sequence states, epigenetic states can be passed from mother to daughter cells during mitosis, and can even persist through meiosis to be transmitted from one generation to the next. Although epigenetic marks can change and revert to their original state far more readily than changes in DNA sequence, they are as fundamental to development and disease as the nature of the DNA sequences on which they reside.
Abundant evidence has demonstrated the importance of epigenetic regulation to human disease—notably cancer. Early observations linked perturbations in DNA methylation to the development of human colorectal cancer and subsequent studies showed that experimental manipulation of DNA methylation state, pharmacologically or genetically, altered tumor development. These have motivated studies that correlate epigenomic profiling of tumor specimens with disease state and clinical outcomes of the individuals providing the specimens. Importantly, ongoing clinical trials using drugs that modify epigenetic states have shown therapeutic promise and the ability to attenuate epigenetic biomarkers that indicate poor prognoses. Although changes in DNA methylation during cancer development and progression have attracted considerable attention, DNA methylation states are also influenced by alterations in histone modification state and nucleosome positioning. The reciprocal interactions among all these epigenetic marks are all likely to be of importance to cancer development and progression.
In addition to controlling processes fundamental to cancer, epigenetic states influence responses of mammals to changes in their environment. Maternal behavior during nursing, exposure to endocrine disruptors and the nutrient composition of diets each have been shown to elicit specific phenotypes that correlate with specific changes in epigenetic states. Most importantly, these phenotypes, and the accompanying epigenetic alterations, can be transmitted from parent to offspring, even if only the parents and not the offspring experienced the environmental insult. This raises the possibility that some complex traits that run in families, like obesity, cancer or behavioral patterns, are transmitted by epigenetic means and result from environmental exposures experienced during prior generations.
A common theme in biology is that mechanisms influencing disease states like cancer, or homeostatic responses to environment are also fundamental to development. Epigenetic mechanisms have been shown to be critical to Drosophila development and mammalian development.
Existing approaches for analyzing epigenetic modifications of chromatin, such as chromatin immunoprecipitation (ChIP), can evaluate only one epigenetic mark at a time and are labor-intensive, serial processes that impose significant limitations on analysis throughput and sample quantity. The ChIP technique involves immunoprecipitation using an antibody specific to one epigenetic modification of interest to isolate modified chromatin, which is subsequently analyzed using massively parallel DNA sequencing, microarray hybridization or gene-specific PCR. This method can be used to characterize the genome placement of a chromatin associated protein and is the predominant analytical tool currently practiced in epigenomic and chromatin research. However, it suffers from two major limitations. First, the analysis requires 104 to 107 cells and is incapable of assessing epigenetic changes in vanishing quantities, making studies of developing embryos, sorted cells or microdissected cells impossible. Second, only one epigenetic mark can be isolated at a time, making detection of co-existent marks very difficult. Measurements provide only information about the ‘average’ chromatin state in a cell population and nothing about the individual DNA strands. In one published example involving the characterization of bivalent states in ES cells, these limitations introduced ambiguity as to if tri-methylated histone H3 lysine 27 (H3K27me3) and tri-methylated histone H3 lysine 4 (H3K4me3) marks were present simultaneously on a given gene or if two populations existed with the ES culture, each with a mutually exclusive mark. Sequential ChIPs against two different antibodies may circumvent this limitation, however this solution is impractical for whole genome analysis.
As such, current methods for epigenomic testing involve bulk molecule analysis. The results are representative of a composite signal from multiple copies of a genetic material, which is distinct from single molecule analysis. Histone modifications are most commonly detected using chromatin immunoprecipitation (ChIP), in which modification-specific antibodies are used to immunoprecipitate the associated DNA, which is then detected by hybridization to microarray (Ren et al. (2000) Science, 290, 2306-2309) (ChIP-chip) or deep sequencing (Barski et al. (2007) Cell, 129, 823-837) (ChIP-seq). DNA methylation can also be detected by immunoprecipitation using a methylcytosine antibody (Weber et al. (2005) Nature Genetics, 37, 853-862), or with bisulfite sequencing, which offers more comprehensive analysis of DNA methylation states (Zhang et al. (2006) Cell, 126, 1189-1201). Genome wide epigenomic analyses using antibodies often use on the order of 106 to 107 cells. ChIP has been used with as few as 100 cells, however, with this few cells the analysis was locus specific and not genome wide (O'Neill et al. (2006) Nature Genetics, 38, 835-841). A far more significant limitation is encountered when studies seek to determine whether or not two or more epigenetic marks are coincident within the genome or are present on a single piece of genetic material, e.g., a single chromatin. Analysis of each epigenetic mark requires an independent immunoprecipitation. When precipitating chromatin from an ensemble of cells with different antibodies, it is difficult to distinguish true coincidence of the detected marks from the existence of multiple populations within the ensemble, each with a different epigenomic profile. This can be somewhat overcome with sequential ChIP, where the material precipitated by one antibody is re-ChIPed with a second antibody (Bernstein et al. (2006) Cell, 125, 315-326). However, these techniques are not amenable to genome wide analysis or for studies in which more than two epigenetic marks are investigated. Furthermore, bulk analysis techniques report an average of the population and do not consider variations at the single molecule level. Thus, there remains a considerable need for alternative methods and compositions that provide for a more robust genome-wide epigenetic analysis. The present invention satisfies this need and provides related advantages.